Conditions of dementia such as Alzheimer's disease (AD) are frequently characterised by a progressive accumulation of intracellular and/or extracellular deposits of proteinaceous structures such as β-amyloid plaques and neurofibrillary tangles in the brains of affected patients. The appearance of these lesions largely correlates with pathological neurofibrillary degeneration and brain atrophy, as well as with cognitive impairment (Mukaetova-Ladinska, E. B. et al. (2000) Am. J. Pathol. Vol. 157, No. 2, 623-636).
Both neuritic plaques and neurofibrillary tangles contain paired helical filaments (PHFs), of which a major constituent is the microtubule-associated protein tau (Wischik et al. (1988) PNAS USA 85, 4506). Plaques also contain extracellular β-amyloid fibrils derived from the abnormal processing of amyloid precursor protein (APP; Kang et al. (1987) Nature 325, 733). An article by Wischik et al. (in “Neurobiology of Alzheimer's Disease”, 2nd Edition (2000) Eds. Dawbarn, D. and Allen, S. J., The Molecular and Cellular Neurobiology Series, Bios Scientific Publishers, Oxford) discusses in detail the putative role of tau protein in the pathogenesis of neurodegenerative dementias.
Studies of Alzheimer's disease indicate that the loss of the normal form of tau (Mukaetova-Ladinska et al. (1993) Am. J. Pathol., 143, 565; Wischik et al. (1995a) Neurobiol. Ageing, 16: 409; Lai et al. (1995b) Neurobiol. Ageing, 16: 433), accumulation of pathological PHFs (Mukaetova-Ladinska et al. (1993), loc. cit.; Harrington et al. (1994a) Dementia, 5, 215; Harrington et al. (1994b) Am. J. Pathol., 145, 1472; Wischik et al., (1995a), loc. cit.) and loss of synapses in the mid-frontal cortex (Terry et al. (1991) Ann. Neurol., 30, 572) correlate with associated cognitive impairment. Furthermore, loss of synapses (Terry et al., loc. cit.) and loss of pyramidal cells (Bondareff et al. (1993) Arch. Gen. Psychiatry, 50: 350) both correlate with morphometric measures of tau-reactive neurofibrillary pathology, which parallels, at a molecular level, an almost total redistribution of the tau protein pool from a soluble to a polymerised form (PHFs) in Alzheimer's disease (Mukaetova-Ladinska et al. (1993), loc. cit.; Lai et al. (1995), loc. cit.).
Tau exists in alternatively-spliced isoforms, which contain three or four copies of a repeat sequence corresponding to the microtubule-binding domain (Goedert, M., et al. (1989) EMBO J. 8, 393-399; Goedert, M., et al. (1989) Neuron 3, 519-526). Tau in PHFs is proteolytically processed to a core domain (Wischik, C. M., et al. (1988) Proc. Natl. Acad. Sci. USA 85, 4884-4888; Wischik et al. PNAS USA 1988, 85:4506-4510); Novak, M., et al. (1993) EMBO J. 12, 365-370) which is composed of a phase-shifted version of the repeat domain; only three repeats are involved in the stable tau-tau interaction (Jakes, R., et al. (1991) EMBO J. 10, 2725-2729). Once formed, PHF-like tau aggregates act as seeds for the further capture and provide a template for proteolytic processing of full-length tau protein (Wischik et al. 1996 Proc Natl Acad Sci USA 93, 11213-11218).
In the course of their formation and accumulation, paired helical filaments (PHFs) first assemble to form amorphous aggregates within the cytoplasm, probably from early tau oligomers which become truncated prior to, or in the course of, PHF assembly (Mena, R., et al. (1995) Acta Neuropathol. 89, 50-56; Mena, R., et al. (1996) Acta Neuropathol. 91, 633-641). These filaments then go on to form classical intracellular neurofibrillary tangles. In this state, the
PHFs consist of a core of truncated tau and a fuzzy outer coat containing full-length tau (Wischik, C. M., et al. (1996b) in “Microtubule-associated proteins: modifications in disease”, eds. Avila, J., Brandt, R. and Kosik, K. S. (Harwood Academic Publishers, Amsterdam) pp.185-241)). The assembly process is exponential, consuming the cellular pool of normal functional tau and inducing new tau synthesis to make up the deficit (Lai, R. Y. K., et al., (1995), Neurobiology of Ageing, Vol. 16, No. 3, 433-445). Eventually, functional impairment of the neurone progresses to the point of cell death, leaving behind an extracellular tangle. Cell death is highly correlated with the number of extracellular tangles (Wischik et al. 2000, loc.cit). As tangles are extruded into the extracellular space, there is progressive loss of the fuzzy outer coat of the neurone with corresponding loss of N-terminal tau immunoreactivity, but preservation of tau immunoreactivity associated with the PHF core (FIG. 4a; also Bondareff, W. et al., (1994) J. Neuropath. Exper. Neurol., Vol. 53, No. 2, 158-164).
The phase shift which is observed in the repeat domain of tau incorporated into PHFs suggests that the repeat domain undergoes an induced conformational change during incorporation into the filament. During the onset of Alzheimer's disease, it is envisaged that this conformational change could be initiated by the binding of tau to a pathological substrate, such as damaged or mutated membrane proteins (see FIG. 19a—also Wischik, C. M., et al. (1997) in “Microtubule-associated proteins: modifications in disease”, eds.
Avila, J., Brandt, R. and Kosik, K. S. (Harwood Academic Publishers, Amsterdam) pp.185-241).
In the case of Alzheimer's disease, current pharmaceutical therapies are focused on symptomatic treatment of the loss of cholinergic transmission which results from neurodegeneration (Mayeux, R., et al. (1999) New Eng. J. Med. 341, 1670-1679). However, although the available treatments delay progression of the disease for up to six to eight months, they do not prevent it. The discovery of drugs that could prevent the aggregation of tau which leads to neurodegeneration would provide a more effective strategy for prophylaxis or for inhibiting the progression of the disease, which would not require an immediate knowledge of the diverse upstream events that initiate the aggregation (FIG. 19b)
Protein Aggregation Assays
Based on the putative model described above, WO 96/30766 describes an in vitro assay for tau aggregation in which a fragment of tau corresponding to the core repeat domain, which has been adsorbed to a solid phase substrate, is able to capture soluble full-length tau and bind tau with high affinity. This association confers stability against proteolytic digestion of the aggregated tau molecules. The process is self-propagating, and can be blocked selectively by prototype pharmaceutical agents (Wischik, C. M., et al. (1996), loc. cit).
Although the in vitro assay described in WO 96/30766 enables the identification of inhibitors or modulators of tau-tau association, the present inventors have also recognized that cell-based models of Alzheimer's disease-like protein aggregation would be useful. Such cellular models could be used both in the primary screening of candidate modulators of tau-tau aggregation, and in the secondary screening of compounds already identified in the in vitro assay of WO 96/30766. Furthermore, the demonstration of tau aggregation in cells could also aid in the identification of normal cellular substrates which are involved in the initiation of pathological tau aggregation, which substrates could themselves be targets for pharmaceutical intervention.
However, numerous papers reporting the expression of various tau constructs in tissue culture models have failed to demonstrate aggregation (see e.g. Baum, L. et al., (1995) Mol. Brain Res. 34:1-17).
These problems can be understood when it is considered that aggregation of soluble tau in vitro has so far only been achieved under non-physiological conditions and at high concentrations (reviewed in Wischik (2000), loc. cit).
Wo 96/30766 also describes two approaches for studying tau aggregation in a cellular environment. In the first approach, full-length tau or fragments of tau were stably expressed in cells. In the second approach, aggregated tau was transiently transfected into cells by use of lipofectin.
Although both of these approaches are useful for the study of tau-tau aggregation, they have some limitations. Transfection of aggregated tau into cells using lipofection is of variable efficiency, as is the production of aggregated tau itself. Moreover, the core tau fragment, which is the most efficient seed for tau aggregation, is found to be toxic when stably expressed in cells, leading to low expression levels. Thus, constitutive expression of the truncated tau fragment of the PHF core in eukaryotic cells is difficult to achieve. Transient expression systems permit the optimization of expression of tau, but the inherent toxicity of the fragments renders even these systems unreliable. Longer fragments of tau are less toxic, but these do not reliably aggregate when expressed in cells.
Thus it would be desirable for an alternative model system to be developed, in which the interaction between e.g. tau molecules and the like could be investigated under physiological conditions, in a stable and controllable cell line, and which could be used to screen for potential diagnostic, prognostic or therapeutic agents of conditions such as Alzheimer's disease.
Animal Models
Although in vitro and cell-based assays can be useful tools, animal models of Alzheimer's disease and related conditions can help to understand the relationship between the biochemical and pathological changes in the brain and impairment of memory and behavior. Furthermore, they enable the pathogenesis of the disease process to be examined in vivo and provide a model in which therapeutic strategies can be tested.
Specifically, animal models put cell-based systems in a pharmacokinetic context where, for example, potential therapeutics can be assessed not just in terms of particular biochemical activities against protein aggregation, but also in terms of toxicity, delivery and half-life at the site of action e.g. through the blood brain barrier.
Notwithstanding this, existing models fail to display the combination of tangles, plaques and cognitive impairment characteristic of AD. Aged dogs and non-human primates develop β-amyloidosis, but tau pathology is not a feature in these animals (Walker, 1997). Similarly, transgenic mice modeling amyloidosis have been created, but these fail to exhibit abnormal deposition of tau (Janus, Chishti, & Westaway, 2000). Conversely animal models of tau pathology fail to demonstrate amyloid pathology. The tangles that accumulate in the brains of rodents treated with aluminium differ in their ultrastructure from those found in AD (see above). Filamentous cytoskeletal changes associated with abnormally phosphorylated tau have been observed in aged baboons, bears, sheep and goats (Braak, Braak, & Strothjohann, 1994; Cork et al., 1988; Nelson, Greenberg, & Saper, 1994; Nelson & Saper, 1995; Roertgen et al., 1996; Schultz et al., 2000).
Some experiments have implicated links between tau and amyloid in animal models. Hyperphosphorylated tau accumulates in the somatodendritic compartment of neurons in rat brains following chronic intraventricular infusion of okadaic acid, an inhibitor of protein phosphatase 2A (Arendt, Holzer, Fruth, Brijckner, & Gartner, 1995). Furthermore, the okadaic acid treatment also led to the formation of extracellular deposits of Aβ and memory impairment. Two other studies have implicated Aβ in the accumulation of tau in animal models. Microinjections of fibrillar Aβ in the cortex of aged rhesus monkeys caused the focal accumulations of intracellular phosphorylated tau (Geula et al., 1998). This was dependent upon both age and species; the same result was not observed for rats or young rhesus monkeys. Secondly, focal deposits of tau have been observed in mice that were transgenic for APP carrying AD associated mutations (Sturchler-Pierrat et al., 1997).
The brains of tau-deficient mice appear immunohistochemically normal and axonal elongation was not affected in cultured neurons (Harada et al., 1994). However, microtubule stability was decreased and its organization altered in some small-calibre axons. Furthermore, an increase in microtubule-associated protein 1A (MAP 1A), which might compensate for a functional loss of tau in large-calibre axons, was found. Thus, tau seems to be crucial in the stabilization and organization of axonal microtubules in certain axons. Subsequent studies have demonstrated that tau-deficient mice exhibit signs similar to certain symptoms characteristic of frontotemporal dementia patients, i.e. personality changes (disinhibition/aggression) and deterioration of memory and executive function. The mice showed muscle weakness and impaired balance control, hyperactivity in a novel environment, and impairment in contextual fear conditioning (Ikegami, Harada, & Hirokawa, 2000). Spatial learning tasks, however, were unaffected in the mice as is the memory function in FTDP-17 patients.
Although neurofibrillary tangles and phenotypic alterations were not reported in animals transgenic for 3- or 4-repeat tau isoforms (Brion, Tremp, & Octave, 1999; Gotz et al., 1995), these findings may reflect low levels of protein expression.
Impaired motor function, in the presence or absence of tauopathy, has been observed in mice expressing higher levels of human tau protein (Ishihara et al., 1999; Spittaels et al., 1999). In these animals the filamentous inclusions did not exhibit the ultrastructural features of AD PHFs.
A recent transgenic mouse model expressing human tau with the P301L mutation develops neurofibrillary tangles, neuronal loss and motor dysfunction (Lewis et at., 2000). The tau inclusions in these mice show both straight and twisted ribbon filaments similar to those found in human patients. Attempts to combine this model with amyloidosis are underway by crossing these mice with those transgenic for APP. Other transgenic mouse models are discussed in U.S. Pat. Nos. 5,912,410 and 5,898,094
Thus it can be seen that existing animal models fail to demonstrate any pathological evidence that the transgene leads to the accumulation of truncated tau protein encompassing the PHF domain. Although filaments are observed in tau carrying the P301L mutation, this corresponds to a form of tau that causes FTDP-17 and not AD. To date, no mutation in the tau gene has been identified which causes AD, and therefore approaches based purely on mutated protein for effect may be of limited relevance.
More particularly, there has been no clear disclosure of unmutated tau aggregating in these models, as evidenced by proteolytic processing to a truncated core fragment corresponding to that found in the actual AD PHF core. A system in which such proteolytic processing occurred in a pharmacokinetic context would thus provide a contribution to the art.